Neuronal RNA binding proteins regulate brain formation - PROJECT SUMMARY The IGF2BP family of RNA binding proteins were recently shown to interact with multiple ASD-associated transcripts and proteins, suggesting it is part of a convergent ASD pathway. However, the fundamental role of IGF2BPs in brain development is poorly defined and is the focus of this proposal. Here we report the discovery of new missense variants for IGF2BP3 found in patients with neurodevelopmental disorders, and phenotype modeling in Drosophila is used to study their function. Loss of the fly IGF2BP3 orthologue Imp from post-mitotic neurons causes microcephaly, and these phenotypes are rescued by wildtype human IGF2BP3 but not patient- associated variants. Imp is known to promote stem cell division, so the finding that Imp has essential functions in, and causes microcephaly when depleted from, non-dividing post-mitotic neurons is novel and surprising yet critical to our understanding of human disease throughout life. IGF2BP1-3 and Imp are RNA-binding proteins that regulate many mRNA targets by modifying stability, transport, splicing, or protein translation. Sap47 (Synapse associate protein 47) is one such target as loss of Sap47 from postmitotic neurons causes microcephaly in flies just like loss of Imp. Sap47 localizes to synapses, but its function in neurons remains largely unknown. Moreover, its mammalian orthologue SYAP1 is a strong ASD risk factor but its function in the mammalian brain is also completely unknown. Imp and Sap47 adversely affect both neuron cell survival and morphology, which likely contribute to microcephaly and a miswired brain characteristic of multi-factorial neurodevelopmental disorders. This proposal will test the hypothesis that IGF2BP3/Imp stabilizes Sap47/SYAP1 mRNA in neurons to promote neuronal targeting and survival and thereby ensure proper brain development. A combination of fly and mouse models as well as human gene variants will be used to investigate the conservation of protein function. First, how Imp and Sap47 loss results in defects in neuron outgrowth, targeting, and survival defects will be investigated using highly characterized fly visual system neurons and mouse hippocampal cultures. The molecular mechanism by which Imp/IGF2BP1-3 regulates Sap47/Syap1 mRNA in neurons will be determined by testing Imp’s role in mRNA stability. Pulse-chase in vivo 5-ethynyluridine (EU) incorporation assays will test Sap47 mRNA stability with and without Imp. IGF2BP3 and patient variants will also be tested. Finally, the mechanism by which Sap47 ensures proper brain development and function will be elucidated using domain analysis. In addition, work here will determine whether loss of putative Sap47 protein interactors cause brain volume phenotypes or modify Imp and Sap47 phenotypes in neurons. Mammalian cell culture will be used to test molecular and biochemical conservation of identified interactions.
